Investigation of the value of precipitins in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) patients with a positive marker for Aspergillus species.

Although a high prevalence of COVID-19-associated pulmonary aspergillosis has been reported, it is still difficult to distinguish between colonization with Aspergillus fumigatus and infection. Concomitantly, similarities between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and hypersensitivity pneumonitis were suggested. The objective of this study was to investigate retrospectively if precipitin assays targeting A. fumigatus could have been useful in the management of SARS-CoV-2 patients hospitalized in an Intensive Care Unit (ICU) in 2020. SARS-CoV-2 ICU patients were screened for Aspergillus co-infection using biomarkers (galactomannan antigen, qPCR) and culture of respiratory samples (tracheal aspirates and bronchoalveolar lavage). For all these patients, clinical data, ICU characteristics and microbial results were collected. Electrosyneresis assays were performed using commercial A. fumigatus somatic and metabolic antigens. ELISA were performed using in-house A. fumigatus purified antigen and recombinant antigens.Our study population consisted of 65 predominantly male patients, with a median ICU stay of 22 days, and a global survival rate of 62%. Thirty-five patients had at least one positive marker for Aspergillus species detection. The number of arcs obtained by electrosyneresis using the somatic A. fumigatus antigen was significantly higher for these 35 SARS-CoV-2 ICU patients (P 0.01, Welch's t-test). Our study showed that SARS-CoV-2 ICU patients with a positive marker for Aspergillus species detection more often presented precipitins towards A. fumigatus. Serology assays could be an additional tool to assess the clinical relevance of the Aspergillus species in respiratory samples of SARS-CoV-2 ICU patients. LAY SUMMARY: This study showed retrospectively that precipitin assays, such as electrosyneresis, could be helpful to distinguish between colonization and infection with Aspergillus fumigatus during the management of severe acute respiratory syndrome Coronavirus-2 (SARS CoV-2) patients in an intensive care unit.

Azole-resistant Aspergillus fumigatus: A global phenomenon originating in the environment?

Aspergillus fumigatus is the predominant etiological agent of invasive aspergillosis (IA), a difficult-to-manage fungal disease associated with a high case fatality rate. Azole antifungals, particularly voriconazole, have significantly improved the survival rate of patients with IA. However, the clinical advances made possible through the use of medical azoles could be threatened by the emergence of azole-resistant strains which has been reported in an ever-increasing number of countries over the last 10 years. The major resistance mechanism, that combines point mutation(s) in the coding sequence of cyp51A gene and an insertion of a tandem repeat in the promoter region of this gene which leads to its overexpression (TR34/L98H and TR46/Y121F/T289A), is presumed to be of environmental origin. However, the emergence of clinical and environmental azole-resistant strains without the cyp51A gene mutation suggests that other mechanisms could also be responsible for azole resistance (for example, overexpression of efflux pumps). The development of resistance may be linked to either long-term use of azole antifungals in patients with chronic aspergillosis (patient-acquired route) or selection pressure of the fungicides in the environment (environmental route). The fungicide-driven route could be responsible for resistance in azole-naive patients with IA. This literature review aims to summarize recent findings, focusing on the current situation of azole-resistance in A. fumigatus, and provides better understanding of the importance of the environmental route in resistance acquisition.

[A case of farmer's lung associated with allergic bronchopulmonary aspergillosis]. / L'association inhabituelle de deux maladies pulmonaires immunoallergiques : un cas de pneumopathie d'hypersensibilité associée à une aspergillose bronchopulmonaire allergique.

Hypersensitivity pneumonitis and allergic bronchopulmonary aspergillosis are two forms of lung disease with presumed distinct immunoallergic mechanisms. We report the observation of a 38-year-old French farmer who, for one month, had fever and dyspnoea resistant to antibiotic therapy. A diagnosis of farmer's lung, and allergic bronchopulmonary aspergillosis was made on clinical, biological, functional and radiological evidence and according to the criteria established. The evolution was favorable with antigenic eviction and corticosteroid therapy. This observation is to our knowledge the 5th case that describes the association of hypersensitivity pneumonitis and allergic bronchopulmonary aspergillosis. It suggests the existence of risk factors and immunoallergic mechanisms common to both diseases and discusses the hypothesis that the same antigen(s) is (are) responsible for them.

Hypersensitivity pneumonitis in a cystic fibrosis patient.

Hypersensitivity pneumonitis (HP) is a chronic inflammatory lung disease caused by repeated inhalation of antigenic substances. We present a case of metalworking fluids (MWFs)-HP sensitized to Pseudomonas oleovorans in a cystic fibrosis patient. This case illustrates that HP diagnosis remains challenging, especially in patients with another pulmonary disease, and that serodiagnosis contributes to identifying the precise microorganism involved. It also demonstrates that P. oleovorans is an important secondary aetiological agent in MWF-HP, less known than Mycobacterium immunogenum.

Identifying indoor air Penicillium species: a challenge for allergic patients.

PURPOSE: Penicillium is the most common mould isolated in housing. Penicillium chrysogenum is the only species tested by prick test or serology for allergic patients. The American Institute of Medicine has accepted Penicillium as an aetiological agent of rhinitis in children and adults and as an asthma agent in children. However, few studies have identified Penicillium in housing to the species level (354 species). Phenotypic identification is difficult. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) should be an alternative. The aim of this study was (1) to identify the Penicillium species present in dwellings in Eastern France and (2) to evaluate the reliability of MALDI-TOF MS for identification, by comparing it to DNA sequencing and phenotypic identification. METHODOLOGY: Identification to the species level was performed by MALDI-TOF MS on 275 strains isolated from 48 dwellings. These results were compared to beta-tubulin gene sequencing and to the phenotypic aspects. RESULTS: Thanks to MALDI-TOF, 235/275 strains could be identified (85.5 %). Fourteen species were identified among 23 Penicillium species included in the Filamentous Fungi Library 1.0 (Bruker Daltonics). However, 72.2  % of the strains belonged to five main taxa: P. chrysogenum (27.3 %), Penicillium glabrum (22.9 %), Penicilliumcommune (11.3 %), Penicillium brevicompactum (6.5 %) and Penicillium expansum (4.2 %). CONCLUSION: Complete coherence between MALDI-TOF MS and sequence-based identification was found for P. chrysogenum, P. expansum, P. glabrum, Penicillium italicum and Penicillium corylophilum. The main drawback was observed for Penicillium crustosum, which included 21 strains (7.6 %) that could not be identified using MALDI-TOF MS.

[Two cases of hot tub lung disease: Environmental investigations]. / Deux cas de pneumopathie d'hypersensibilité du jacuzzi : enquête environnementale.

INTRODUCTION: Hot tub lung is a hypersensitivity pneumonitis (HP) due to exposure to inhaled non-tuberculous mycobacteria, the most frequent being Mycobacterium avium complex (MAC). CASE REPORT: A French couple developed typicalHP in the context of a repeated use of hot tubs. The husband had a severe hypoxemic form whereas his wife had a micronodular form with patchy ground glass on the thoracic scan, with less severe functional impairment. MAC was recovered in the hot tub water, but not in broncho-alveolar lavage fluid, and serologies were negative. Samples taken at home showed unusual exposure to Aureobasidium pullulans and Aspergillus flavus, as well as the presence of potentially responsible domestic molds. Blood precipitins for these microorganisms were identified. The evolution was favorable after removal of the hot tub. CONCLUSIONS: These cases represent two of the typical presentations of hot tub lung, with a possible HP to an antigen other than MAC, which may have been enhanced by chronic exposure to multiple microorganisms.

Emergence of Aspergillus fumigatus azole resistance in azole-naïve patients with chronic obstructive pulmonary disease and their homes.

Azole-resistant Aspergillus fumigatus (ARAF) has been reported in patients with chronic obstructive pulmonary disease (COPD) but has not been specifically assessed so far. Here, we evaluated ARAF prevalence in azole-naïve COPD patients and their homes, and assessed whether CYP51A mutations were similar in clinical and environmental reservoirs. Sixty respiratory samples from 41 COPD patients with acute exacerbation and environmental samples from 36 of these patient's homes were prospectively collected. A. fumigatus was detected in respiratory samples from 11 of 41 patients (27%) and in 15 of 36 domiciles (42%). Cyp51A sequencing and selection on itraconazole medium of clinical (n = 68) and environmental (n = 48) isolates yielded ARAF detection in 1 of 11 A. fumigatus colonized patients with COPD (9%) and 2 of 15 A. fumigatus-positive patient's homes (13%). The clinical isolate had no CYP51A mutation. Two environmental isolates from two patients harbored TR34 /L98H mutation, and one had an H285Y mutation. Coexistence of different cyp51A genotypes and/or azole resistance profiles was detected in 3 of 8 respiratory and 2 of 10 environmental samples with more than one isolate, confirming the need for a systematic screening of all clinically relevant isolates. The high prevalence of ARAF in patients with COPD and their homes supports the need for further studies to assess the prevalence of azole resistance in patients with Aspergillus diseases in Northern France.

Common proteins located in pigeon, budgerigar, and hen droppings related to bird fancier's lung

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Second hypersensitivity pneumonitis in the same patient caused by chinchillas

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[Classical and molecular methods for identification and quantification of domestic moulds]. / Méthodes d'identification et de quantification des moisissures de l'habitat : méthodes classiques, méthodes moléculaires.

INTRODUCTION: To study the impact of the constant and inevitable inhalation of moulds, it is necessary to sample, identify and count the spores. BACKGROUND: Environmental sampling methods can be separated into three categories: surface sampling is easy to perform but non quantitative, air sampling is easy to calibrate but provides time limited information, and dust sampling which is more representative of long term exposure to moulds. The sampling strategy depends on the objectives (evaluation of the risk of exposure for individuals; quantification of the household contamination; evaluation of the efficacy of remediation). The mould colonies obtained in culture are identified using microscopy, Maldi-TOF, and/or DNA sequencing. VIEWPOINTS: Electrostatic dust collectors are an alternative to older methods for identifying and quantifying household mould spores. They are easy to use and relatively cheap. Colony counting should be progressively replaced by quantitative real-time PCR, which is already validated, while waiting for more standardised high throughput sequencing methods for assessment of mould contamination without technical bias. CONCLUSION: Despite some technical recommendations for obtaining reliable and comparable results, the huge diversity of environmental moulds, the variable quantity of spores inhaled and the association with other allergens (mites, plants) make the evaluation of their impact on human health difficult. Hence there is a need for reliable and generally applicable quantitative methods.

[European birth cohorts: Early life exposure to microorganisms and health impact]. / Cohortes de naissances européennes : exposition aux micro-organismes et impact sanitaire.

In recent years, many birth cohorts have been initiated in Europe, to assess the early life microbiological exposure of children in the indoor environment and better understanding the different effects (adverse/protectors) on health. The results of 12 European cohorts, with different methodologies for exposure and allergic risk assessment are summarized in this review. Four meta-analyzes of cohort are presented too. Microbiological researches in indoor environment seem to turn to a metrology of microbiological exposure, but few studies provide real quantitative data. Thus, the establishment of dose-effect relationship is not possible and can only be done by having a global view of the situation, provided by an identical metrological approach in the different studies, in a large-scale, in the context of large birth cohorts with children followed with strict criteria to establish the clinical diagnosis.

Azole-resistant Aspergillus fumigatus in sawmills of Eastern France.

AIMS: Emergence of azole-resistant Aspergillus fumigatus complicates management of Aspergillus diseases. Currently, selection pressure caused by azole fungicide use in farming is strongly suspected of creating resistance. As sawmills also use azole fungicides, we investigated the presence of azole-resistant strains in this environment and studied the relationship between azole fungicide use and development of resistance. METHODS AND RESULTS: Air (n = 200) and substrate (n = 600) samples were taken in 20 sawmills. Azole-resistant strains (Etest and EUCAST methods) were confirmed by sequencing the cyp51A gene and its promoters. Dosage of propiconazole and tebuconazole was performed by gas chromatography coupled with mass spectrometry. Twenty-four azole-resistant A. fumigatus strains were collected among 20 of the 600 substrate samples (3%). Eighty-three percent of theses strains had TR34 /L98H mutation. A significantly higher number of resistant strains was collected in sawmills using fungicide products made with propiconazole mixed with a high concentration of tebuconazole (P = 0·009). The presence of resistant strains was significantly linked to propiconazole quantities in substrates (P = 0·03). CONCLUSIONS: The outcome of azole-resistant A. fumigatus carrying TR34 /L98H mutation seems to greatly depend on the azole fungicide formulation and quantities of azole. These preliminary results are valuable to propose new approaches limiting the emergence of azole-resistant strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Azole resistance is an emerging problem in A. fumigatus and threatens clinical advances made possible by the use of azole antifungals in the treatment of Aspergillus-related diseases. Azole fungicides are also used in the wood industry, notably in sawmills, to protect wood from wood-destroying fungi. Through our study, we show that sawmills represent another professional environment affected by the presence of azole-resistant A. fumigatus strains carrying the TR34 /L98H mutation. Moreover, this study provides valuable preliminary results to propose some new approaches to limit the emergence of azole-resistant A. fumigatus strains.

Microbiological consequences of indoor composting.

Recycling of organic waste appeals to more and more people. The aim of this study was to evaluate the microbiological contamination around organic waste bins at three distances over a 12-month period. Contamination near the customary trash of control households was evaluated at the beginning to ensure that there is no recruitment bias. Air samples using the MAS 100 impactor were carried out in 38 dwellings that do household waste composting and in 10 dwellings of controls. Collection of particles by CIP 10 rotating cup sampler and dust samples collected by electrostatic dust collector cloths were acquired in dwellings that do household waste composting. Samples were analyzed by culture and by real-time quantitative PCR. Information about dwelling characteristics and inhabitant practices was obtained by a standardized questionnaire. The genera most often isolated were Penicillium, Aspergillus, Cladosporium and Streptomyces. Near the organic waste bins, bioaerosol samples showed an increase of Acarus siro (P = 0.001). Sedimented dust analyses highlighted an increase of A. siro, Wallemia sebi, Aspergillus versicolor, and Cladosporium sphaerospermum concentrations after a 12-month survey compared to the beginning. Composting favors microorganism development over time, but does not seem to have an effect on the bioaerosol levels and the surface microbiota beyond 0.5 m from the waste bin.

[Azole resistance with environmental origin: What alternatives for the future?]. / Résistance aux antifongiques azolés d'origine environnementale : quelles alternatives pour l'avenir ?

Azole resistant Aspergillus fumigatus strains are increasingly reported in many countries. One resistance mechanism is attributed to the use of azole fungicides in environment. Two mutations, TR34/L98H and TR46/Y121F/T289A, on the cyp51A gene, have been described. Results of 40 publications about azole resistant strain detections, with TR34/L98H and TR46/Y121F/T289A mutations, in clinical and/or environmental samples, are presented in this review. These cases, observed in many countries, suggest spreading phenomenon. Measures to moderate fungicides treatments and/or alternative treatments in environment should be established to preserve the effectiveness of azole antifungal therapy for at-risk patients.

Microbiological characterization of 3193 French dwellings of Elfe cohort children.

Although exposure to indoor microorganisms in early life has already been associated with respiratory illness or allergy protection, only a few studies have performed standardized samplings and specific microbial analysis. Moreover, most do not target the different groups of microorganisms involved in respiratory diseases (fungi, bacteria, dust mites). In our study, ten specific qPCR targets (6 fungal species, 1 family and 2 genera of bacteria, 1 house dust mite) were used to analyze the microorganism composition of electrostatic dust fall collector (EDC) from 3193 dwellings of the Elfe French cohort study. Multivariate analyses allowed us to show that the microbial composition of dwellings, assessed with simultaneous analysis of 10 microorganisms, can be characterized by four entities: three bacteria, house dust mite Dermatophagoïdes pteronyssinus, fungi Alternaria alternata, and five other molds. Some dwellings' intrinsic characteristics (occupational ratio, type of dwelling and presence of pets) clearly influence microorganism distribution, and six different profiles of dwellings, characterized by their composition in microorganisms, have been described across France. The use of these clusters seems promising in the evaluation of allergic risk. Allergic respiratory diseases will develop in the near future in some children of the Elfe cohort and will indicate to what extent our approach can be predictive of respiratory disease.

A 10-year survey of fungal aerocontamination in hospital corridors: a reliable sentinel to predict fungal exposure risk?

BACKGROUND: Invasive mould infections represent a threat for high-risk patients hospitalized in haematology units. French guidelines recommend that fungal aerocontamination monitoring should be performed quarterly. Since 2002, Besançon University Hospital has expanded to include several new buildings. Consequently, environmental surveys have been re-inforced and are now performed on a weekly basis. AIM: To retrospectively assess the contribution of fungal aerocontamination measurement in haematology corridors and main hospital corridors as a sentinel to assess fungal exposure and risk of invasive mould infections. METHODS: Over a 10-year period, 2706 air samples were taken by impaction every week in the same locations in haematology corridors and main hospital corridors. All fungal species were identified. The Haematology and Hospital Hygiene Departments were alerted systematically whenever a peak of opportunistic species was detected and corrective action was planned. Since 2007, each case of invasive aspergillosis has been reported to the French health authorities. Cuzick's test, Mann-Kendall's trend test, autocorrelation and Spearman's correlation rank test were used for statistical analysis. FINDINGS: Over 10 years of surveillance, 12 peaks of Aspergillus fumigatus (>40 colony-forming units/m(3)) were observed in the main hospital corridors, and A. fumigatus contamination was detected up to six times per year in the haematology corridors. In order to limit fungal exposure, the decision was made to perform additional checks on ventilation systems and heating, increase biocleaning and develop clear instructions. CONCLUSION: No significant link was observed between A. fumigatus detection and invasive aspergillosis. Weekly surveys have helped to improve the vigilance of the medical teams. Nevertheless, 58 cases of invasive aspergillosis have been identified since 2007.

Evaluation of invasive aspergillosis risk of immunocompromised patients alternatively hospitalized in hematology intensive care unit and at home.

UNLABELLED: Contrary to hospital exposure, little is known about the indoor fungal exposure of hematology patients at home. The aim of our study was to investigate the mold exposure of hematology patients both at home and at hospital to assess their invasive aspergillosis (IA) risk. Fungal exposure was assessed by quantifying opportunistic molds at hospital during hospitalization and in homes of 53 hematology patients. IA was diagnosed in 13 of 53 patients and invasive fungal infection (IFI) in one patient. In hospital, no opportunistic species, or low levels of opportunistic species, were found in 98% of weekly controls. Only 2% of hematology intensive care unit (ICU) controls showed a high level of Aspergillus fumigatus spores in corridor air. Five patients IA were hospitalized during these periods. Seven dwellings of 53 (5/14 dwellings of patients with IA/IFI and 2/39 dwellings of non-IA patients) had a percentage of A. fumigatus and Aspergillus flavus to total mold (significant predictor variable of IA/IFI in our study, general linear model, P-value = 0.02) as high as 15%. Maintaining a 'zero Aspergillus' goal at hospital is essential, and establishing specific and individually opportunistic mold monitoring at home could help to further reduce the IA risk through continuous surveillance. PRACTICAL IMPLICATIONS: This study emphasizes the fact that preventive measures should not be aimed only at the hospital setting: among patients diagnosed with invasive aspergillosis/invasive fungal infection (IA/IFI), 5 of 14 (36%) were exposed to opportunistic fungal species at home exclusively. Moreover, four of these five patients were living in homes having the highest percentage of Aspergillus fumigatus and Aspergillus flavus (>15%), one of which had 48% of A. fumigatus. Therefore, our work supports the need for a counselor to carry out an environmental survey in patients' homes.